DEVELOPMENTAL

BIOLOGY

47, 237-256 (19%)

Lizard Myogenesis in Vitro: A Time-Lapse and Scanning Electron Microscopic Study’ ELLEN

KUHN

BAYNE

AND

SIDNEY

B.

SIMPSON,

JR.

Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201 Accepted June 20,1975 Cultured lizard myoblasts exhibit a distinctive rounded morphology during the G, (G,?) period of the cell cycle immediately preceding cell fusion (Cox, P. G. (1968). J. Morphol. 126, l18). To characterize further these prefusion myoblasts, developing colonies of cultured lizard myogenic cells were studied with the aid of time-lapse cinemicrography and scanning electron microscopy @EM). Time-lapse observations have shown that the postmitotic prefusion myoblast has both a distinctive morphology and a behavior that serve to disinguish it from other myogenic cells within a developing colony. The rounded morphology of these cells, although initially unstable, becomes highly stable once these cells constitute 68-70% of the cell population in a colony. Stable rounded prefusion cells were not observed to undergo cell division. SEM observations of cells in fixed staged myogenic colonies have shown that the rounded prefusion myoblasts in pre-, early, and late aggregation stage colonies exhibit different surface topographies. The different surface topographies are independent of the medium (fusion permissive or nonfusion) that the cells are grown in. This study, although primarily descriptive, serves to define for the first time a discrete set of changes in both cell behavior and cell surface topography exhibited by prefusion myoblasts. INTRODUCTION

The differentiation of multinucleated muscle fibers involves fusion of mononucleated cells (Lash et al., 1957; Konigsberg, 1963; Okazaki and Holtzer, 1966; Mintz and Baker, 1967). Fusion of myoblasts occurs during the G, phase of the cell cycle (Strehler et al., 1961; Okazaki and Holtzer, 1966; Bischoff and Holtzer, 1969; O’Neill and Stockdale, 1972a; among others). Particular significance has been ascribed to the G, period prior to fusion. It has been suggested that this period may be required for the biochemical changes leading to both fusion and the synthesis of characteristic muscle proteins (Okazaki and Holtzer, 1966; Yaffe, 1971;Holtzer and Sanger, 1972). Holtzer and his colleagues (Bischaff and Holtzer, 1969; and reviews by Holtzer and Bischoff, (1970) and Bischoff (1970))have further proposed that the postmitotic prefusion myoblast is the product of a “quanta1 division” as opposed to a “proliferative division” and that the behav’ Supported NIGMS, NIH.

by Grant

No. GM 18269 from

the 237

Copyright 0 1975 by Academic press, Inc. All rights of reproduction in any form reserved.

ior of postmitotic prefusion myoblasts is quite different from that of the parent presumptive myoblast. Recent studies have challenged both the necessity of invoking a “quanta1 mitosis” and the proposed withdrawal of the cells produced by such a mitosis from the proliferative pool (O’Neill and Stockdale, 1972a,b; Stockdale and O’Neill, 1972; Buckley and Konigsberg, 1974; Konigsberg and Buckley, 1974; Doering and Fischman, 1974). Analysis of the putative postmitotic prefusion myoblast ideally requires an experimental system in which this cell possesses a reasonable half-life, can be identified in living cultures, and can be collected in large numbers. Established myogenic cell lines, originally derived from the tail blastema of the lizard, fulfill these requirements. In the lizard system, myoblasts in the G, period of the cell cycle immediately preceding cell fusion assume a distinctive rounded-up morphology (Cox, 1968). These rounded prefusion myoblasts do not appear in cultures until colonies are quite large. They then accumulate in colonies over a period of several days prior to their

238

DEVELOPMENTAL

BIOIOGY

fusing to form multinucleated myotubes if cultured in fusion permissive media, or their differentiation into long, attenuated contractile mononucleated cells if cultured in nonfusion media. Previous studies have shown that these rounded prefusion cells do not incorporate PH]TdR (Cox, 1968; Simpson and Cox, 1972), suggesting strongly that they represent a nonproliferating population. Likewise, Cox and Simpson (1970), utilizing a microspectrophotometric method for measuring the DNA content of nuclei in these rounded prefusion myoblasts, have demonstrated that they are not part of the proliferative pool and are in the G, (G,?) phase of the cell cycle. While these results are consistent with the existence of a postmitotic prefusion myoblast population, they were based on static fixed cultures. In the present communication we present observations based on continuous time-lapse studies that provide additional evidence for the existence of postmitotic prefusion myoblasts in the lizard myogenic system. The time-lapse observations have also enabled us to more accurately define and identify the various morphologies assumed by myogenic cells in developing colonies. With this information in hand we have proceeded to the identification of these morphological cell types at the level of the scanning electron microscope @EM). The SEM observations presented herein relate primarily to changes in surface topography exhibited by the postmitotic prefusion myoblasts. The time-lapse observations have shown that: (1) The postmitotic prefusion myoblast has a distinctive morphology and behavior that serve to distinguish it from other myogenic cells within a developing colony; (2) the rounded-up morphology of these cells, although initially unstable, becomes highly stable once these cells constitute 68-70% of the cell population in a given colony; (3) although many of these cells have been observed continuously for periods of up to 36 hr, none was observed to undergo division; and (4) these cells enter

VOLUME

47.

1975

an active aggregation phase prior to their differentiation into spontaneously contracting muscle cells. SEM observations of cells in fixed staged myogenic colonies have shown that: (1) The rounded postmitotic prefusion myoblasts in preaggregation, early aggregation, and advanced aggregation stage colonies exhibit different surface topographies (2) these different surface topographies are independent of the type of medium (fusion permissive or nonfusion) in which the cells are cultured; and (3) the surface topography of mononucleated and multinucleated myocytes is similar, if not identical. MATERIALS

AND

METHODS

Myogenic cell line. For this study cell lines established from promuscle aggregates of the regenerating tail of the lizard A&is carolinensis were used. Such cell lines have been routinely established from lizard promuscle cells and can be maintained in culture indefinitely when grown in nonfusion media. These lines retain their ability to differentiate into multinucleated contracting muscle cells when cultured in fusion permissive media (Simpson and Cox, 1972). By all available criteria these lines are not transformed; they retain the morphology and behavior of the original primary cultures; they are easily established; they do not exhibit senescence; and they do not give rise to tumors when injected into immune suppressed lizards (Simpson and Cox, 1972). Most of the work described utilized cell line M 23SlO which had been carried in culture for 7 months. Our studies also included observation of two older cell lines. No differences were noted between the three cell lines. The scanning micrographs presented in this paper are based on the studies of cell line M 23SlO. SEM. Cells were plated in 60-mm Falcon plastic dishes each containing several pieces of Thermanox coverslip. Initial plating densities were 103or 10“ cells per dish. Coverslip pieces with attached cells were

BAYNE AND SIMPSON

transferred to 35mm dishes 1 day after plating and cells were maintained in nonfusion or fusion permissive medium as previously described (Cox, 1968; Simpson and Cox, 1972). At appropriate stages, cells attached to coverslips were rinsed gently in phosphate-buffered saline (PBS), pH 7, and fixed in 2% glutaraldehyde in phosphate buffer, pH 7. The glutaraldehyde solution, added slowly (drop by drop) to the last change of PBS was replaced by fresh glutaraldehyde fixative. During PBS rinses and subsequent fixation the cells were observed with an inverted phase microscope to assure that treatment did not result in retraction of cells from the substratum. Cells were postfixed in 1% 0~0, in phosphate buffer, dehydrated in increasing concentrations of ethanol and rinsed with Freon transitional fluid for critical point drying in a Bowmar apparatus. Preparations were coated with thin layers of carbon and gold and viewed in a Kent Cambridge SEM at 20 kV. Time lapse. Cells were plated at 103 or 104 per 60-mm Falcon plastic dish. Between day 8 and 10 of culture, dishes containing centrally positioned colonies were chosen for photography. The dishes were capped with the tops from Falcon Cooper dishes. Sterile stopcock grease was used to seal the cultures. A colony in which l&20% of the central cells exhibited the rounded prefusion morphology was chosen for cinemicrography. Time-lapse sequences ranging from 24 hr to 7.7 days were made. A Wild M 20 phase microscope fitted with a Bolex camera, driven by a Sage time-lapse apparatus was used. The cultures were maintained at 30-31°C with a Sage air curtain incubator. Framing rates ranged from 1 to 2 frames/min. Magnification was 19 x. Media

Two media were employed in these studies. Nonfusion or growth medium: Ham’s F-10, 10% horse serum, 5% chick embryo extract, and 1% GIBCO antibiotic-antimy-

Lizard

239

Myogenesis

cotic mixture. Fusion medium: Eagle’s MEM with the same added biologicals. RESULTS

Time-Lapse

Studies

The following sequence describes the changes in morphology and behavior of cells in a myogenic colony from a lo-day culture (grown in nonfusion medium) during a 7.7-day time-lapse sequence. Additional filmed sequences of shorter duration, focused on specific cell types, have been analyzed to augment the continuous sequence. Filming was initiated when the colony consisted of l&20% rounded prefusion myoblasts. Choosing a colony at this stage allows positive identification of a myogenic colony. Since the rounded prefusion cells first appear centrally within the colony, an area midway between the central rounded cells and the periphery of the colony was chosen for photography. This area was still mitotically active at the start of the sequence. Thus, by photographing a small window between center and periphery of the colony one can record the progression of colony differentiation as it develops from center to periphery. Since the film sequence was initiated in a lo-day culture, the time points (days) of the time-lapse sequence (Fig. 1) are not to be confused with the days of culture. The results of the time-lapse study confirm and extend previously published descriptions of colony differentiation (Cox, 1968; Simpson and Cox, 1972) that were based on series of fixed cultures. The time-lapse studies have also provided information on cell behavior that could not be derived or inferred from fixed cultures. The sequence of changes during the 7.7day time-lapse sequence is illustrated in Fig. 1. Initially the cells of a colony are either flattened onto the dish and appear “fibroblastlike” or they are plump bipolar cells, (Fig. 1A). The small number of cells with a rounded morphology seen in Fig. 1A represent mitotic cells. All of the

FIG. 1. Sequence of frames, A-H, from a 7.7-day time-lapse movie. Numbers in lower left hand corner of each frame give time in days. Initially most of the cells have a flattened or bipolar morphology (A); with time the rounded G, prefusion cells make their appearance (B); the rounded cells increase in number (C-D); the rounded cells begin forming loose aggregates (E-F); the rounded cells begin to stretch back onto the culture dish (G-H). Rounded cells accumulate within a colony until roughly 70% of the population is rounded (A, 7.6%; B, 22.8%; C, 37.8%; D, 60.1%; E, 69.5%; F, 68.9%; G, 61.0%; H, 69.8%); r, rounded cell; a, aggregate of rounded cells; s, “stretched-out” cell. X 171

BAYNE

AND

SIMPSON

rounded cells observed during the first 1.6 days of the time-lapse sequence proved to be mitotic cells. These mitotic cells are the result of flattened or bipolar cells that have rounded up in preparation for cell division. They remain in the rounded state for no longer than 15-20 min prior to completion of cytokinesis. The daughter cells resulting from such a division are smaller (5-6 pm) in diameter than the parent metaphase cell (lo-12 pm) and undergo vigorous bubbling activity for a period of 15-20 min before spreading back onto the substratum as flattened or bipolar cells. By 1.9 days into the sequence, the bipolar cells increase in number relative to the flattened cells. At this time a new cell type with a rounded-up morphology and a distinctive behavior pattern first appears. This cell type is the postmitotic prefusion myoblast described by Cox (1968). These myoblasts rounded-up prefusion are smaller (8-9 pm) in diameter than metaphase cells and larger than daughter cells resulting from a mitosis. Unlike metaphase or anaphase cells they are attached to the substratum via flattened sheetlike extensions or long stalklike lobopodia, both of which exhibit ruffled membranes. These rounded-up prefusion cells, unlike the rounded mitotic cells, exhibit directed locomotory behavior and remain roundedup for extended periods of time. Many of them have been followed for up to 36 hr, prior to their exiting from the field of view. Of particular interest to us was the observation that some of the chronologically younger rounded-up prefusion cells (Fig. lB-D) exhibit a period of morphological instability. They often vacillate between the characteristic rounded-up morphology and a plump bipolar morphology. After reversing morphology several times they ultimately stabilize in the rounded-up form. With time rounded cells accumulate until they constitute 68-70% of the cell population (Fig. lE-H). Once this point is reached, the rounded-up morphology is stable. The number of rounded prefusion cells

Lizard

Myogenesis

241

exhibiting the stable morphology progressively increased from 2.9 to 4.7 days in the filmed sequence. Thus it would appear that the increase in numbers of roundedup prefusion cells with time, described in previous studies (see Simpson and Cox (1972) for review) is the result of two factors. First, the increase is due to increasing numbers of cells leaving the mitotic pool and entering an extended G, period. Second, the increase is in part due to progressive stabilization of the rounded-up morphology. The stable rounded-up prefusion cells recorded in the time-lapse sequence were never observed to undergo mitosis. Only flattened or bipolar cells engaged in cell division. The mitotic activity within the filming field progressively decreased. Between initiation of filming and 1.9 days, 32 mitotic events were recorded. Thirty-nine mitotic events were observed between day 2.9 and 3.4. Twenty-six mitoses occurred between day 3.5 and 4.7, while only 14 mitoses were observed between day 4.8 and 7.7. The low mitotic activity seen in the last 3 days of the time-lapse sequence is also characteristic of entire colonies where the number of rounded-up cells approaches 70%. Counts of mitotic figures (metaphase, anaphase, and telophase) were made using fixed and stained colonies in which 40-70% of the cells were rounded-up prefusion myoblasts. The percentage of mitotic cells in these colonies ranged from 3.9 to 0.22 with a mean of 1.4%. Thus an accessment of mitotic activity by two independent techniques indicates that in colonies where at least 4070% of the cell population exists as rounded-up prefusion cells, the percentage of cells undergoing mitosis is very low. It thus follows that the chances of mistaking a mitotic cell for a rounded-up prefusion cell are low in colonies at this stage of development. Further, the low mitotic activity emphasizes the improbability of rounded prefusion myoblasts in such colonies undergoing division.

242

DEVELOPMENTAL BIOIX)GY

While the latter points may seem trivial, they have either been insufficiently emphasized in previous publications (Cox, 1968; Simpson and Cox, 1972) or misunderstood by many workers unfamiliar with the lizard system. The first incipient signs of aggregation were seen as the proportion of stable rounded-up prefusion cells approached 60% of the cell population. Prior to this time the majority of the contacts between rounded prefusion cells resulted in ruffled membrane paralysis and the cells moving away from each other. Here however, many of these cells contact each other, remain juxtaposed, and move in concert. The first loose clumps or aggregates of rounded-up cells were observed on day 3 of the time-lapse sequence (Fig. 1D). Aggregates composed of three to four cells moved as a unit, frequently coming together to form larger aggregates. Large aggregates (lo-20 cells) had formed by 6.5 days into the sequence. These aggregates were multilayered and changed in size and shape with time. The aggregates appeared to be rather loosely arranged. Small clusters of cells occasionally broke away and other groups of cells

VOLUME 47, 1975

were added to the main aggregate. There appeared to be considerable movement of cells within the large aggregates. Near the end of the 7.7-day film sequence a number of the rounded-up cells within aggregates were stretching back onto the surface of the dish as the typical 150-200~pm stretched out cells (Figs. 1G and H). These are the cells that will later become spontaneously contracting mononucleated muscle cells. SEM Studies

Myogenic colonies observed in the SEM were staged (Table 1) on the basis of (1) percentage of rounded-up cells; (2) extent of aggregation as well as size of aggregates; and (3) relative numbers of stretched back (mononucleated muscle cells) in the older cultures grown in nonfusion media. Cultures either grown in fusion permissive medium or switched to fusion medium provided colonies with large numbers of multinucleated myotubes. Although the majority of the myogenic colonies in a culture dish of a given chronological age are roughly at the same stage with regard to any of the criteria listed above, a number of smaller secondary colonies are

TABLE

1

CRITERIA FOR STAGING LIZARD MYOGENIC COLONIES” Stage

Preaggregation Early aggregation Late aggregation Stretched out FusionC

Age of cultureb (days)

Rounded prefusion myoblasts (o/o)

Relative number of aggregates

Largest aggregate size (No. of cells)

Rela~~o~m-

10-12 13-15

[lo-501 [60-701

L-1 [+I

WI

-

-

15-20

[60-701

[+++I

[a501

(+I

-

18-21 23

[25-401 [lo-201

[+++I [+I

[-Id [+I’

stretched out mononucleated muscle cells

a Based on cultures plated at 103 cells per 60-mm Falcon dish in nonfusion medium. bracketed parameters are diagnostic for each stage. b Culture age is of general rather than diagnostic value in staging colonies. c Cultures grown in nonfusion medium and switched to fusion medium on day 21. * Less than 5% of nuclei in myotubes. e Sixty-five percent of nuclei in myotubes.

Multinucleated myotubes

Combinations

of

BAYNE AND SIMPSON

always present in cultures where roundedup cells have formed. These secondary colonies often represent a variety of younger stages in the development of a myogenic colony. Thus in a culture where 80-90% of the colonies are in an advanced aggregation stage, the smaller secondary colonies may have only 30% rounded-up cells and therefore represent a preaggregation stage. Young secondary colonies can even be found in cultures grown in fusion permissive medium, where the majority of the colonies exhibit multinucleated myotubes. These secondary colonies have in each case served as internal controls for the preparative procedure. Since in every case the surface topography of the cells in the secondary colonies was appropriate for their stage of development, we are confident that the stage specific changes in cell surface topography of the G, prefusion myoblasts described below are real and cannot be attributed to variations in fixation and critical-point drying of individual preparations. Although most of the observations to be described are based on colonies in nonfusion medium, cells grown in fusion permissive medium were also examined and exhibited identical surface topographies. Preaggregation stages (nonfusion medium). Colonies at preaggregation stages exhibit varying numbers of rounded prefusion myoblasts. The youngest colonies examined in this study had 10% rounded cells, while the oldest preaggregation colonies had between 30 and 50% rounded cells. The rounded prefusion myoblasts in preaggregation colonists have a diameter of 8-9 pm. They are most frequently attached to the culture dish via flattened sheetlike extensions (Fig. 3) that often exhibit ruffled membranes. Rounded myoblasts supported by a single thick stalk or lobopodium are also seen. These two modes of attachment to the culture dish were seen in the time lapie movies. In the latter, these two attachment morphologies

Lizard

Myogenesis

243

are exhibited at different times by the same rounded myoblast. The cell surface of the rounded myoblast is covered with numerous blebs of varying size (Figs. 2 and 3). In preaggregation colonies where at least 50% of the cells are rounded myoblasts, these rounded cells also exhibit filopodia.2 The filopodia vary in width from 0.15 to 0.26 pm and range up to 26 pm in length. They interconnect adjacent rounded myoblasts (Figs. 2 and 3). They also contact cell types of different morphology and contact the substratum as well. The filopodia are often wider at the base (0.3 pm) and gradually taper to a width of 0.26-0.15 pm. Those that contact the surface of the culture dish usually exhibit a terminal bulb or swelling (Fig. 4B). Examples were observed in which the tip of a filopodium made contact with a surface bleb on an adjacent rounded myoblast (Fig. 4D). Here the tip of the filopodium appeared to have a shallow conical morphology. In some cases the base of a filopodium closely approached the size and morphology of the surrounding surface blebs (Fig. 40. We have observed that rounded myoblasts in the youngest preaggregation colonies (i.e., small colonies with few rounded myoblasts) exhibit filopodia infrequently. 2 Minute projections extending from the surface of cells in tissue culture have been labeled by various authors as: microvilli; microspikes; microextensions; retraction fibrils; and filopodia (Taylor, 1966; Dalen and Scheie, 1969; Follett and Goldman, 1970; Porteretal., 19721973; Shimadaetal., 1974). Inour material we observe extensions that vary in width from 0.15 to 0.26 pm and that vary in length from 1.5 to 26.0 pm. Neither from our own observations nor from those reported in the literature can we find any convincing evidence to argue against the proposition that all of these projections are merely gradations in the expression of a single family of surface moditications. However, some surface projections are considerably longer than others. For descriptive purposes we have arbitrarily defined microvilli as thin surface projections that are 5 pm or less in length. Likewise, thin surface projections that range from 6 to 26 Frn or more in length are defined as filopodia. These designations follow, at least in spirit, those used by Porter et al. (19721.

BAYNE AND SIMPXIN

A second cell type encountered in early preaggregation colonies is a flattened irregularly shaped cell (Fig. 3). The surface topography of these cells is typically very smooth, only occasionally adorned with scattered blebs, clusters of small blebs and variable numbers of microvilli. Another cell type, with a bipolar morphology, occurs in preaggregation as well as early aggregation colonies. Based on surface topography two types of bipolar cells can be distinguished. Type A bipolars (Fig. 4E) have a relatively smooth surface adorned with scattered microvilli and clusters of small blebs. Long filopodial processes are frequently associated with the blebbed patches. A ruffled membrane is often observed at one pole of the Type A bipolars. Type B bipolar cells (Fig. 4F) differ from Type A in that: (1) they tend to be shorter and thicker; (2) they have a distinct ruffled membrane at both ends; and (3) the cell surface is highly blebbed. The number of blebs approaches that seen in the rounded myoblasts. The Type B bipolar cell also exhibits a greater number of filopodia than do the Type A bipolars. These two surface topographies may well represent surface modulations of a single bipolar cell type rather than two distinct cell types. Our study cannot distinguish between these alternatives. However, the gross cell shape of the Type B bipolar cell is most similar to the plump bipolar cells seen in the time lapse sequences. There, the rounded myoblasts were seen to vacillate between the rounded morphology and a plump bipolar morphology. Cells identified as putative mitotic cells were frequently seen in the colonies. In young preaggregation colonies they were found scattered throughout the colony. In older colonies they were most often found

FIG. 2. Typical

preaggregation

Lizard

Myogenesis

24s

near the periphery of the colony. We have provisionally judged them to be mitotic stages based on (1) their size; (2) frequency of occurrence and, (3) their surface topography. Their size and frequency of occurrence correlate with that seen in living colonies of comparable stage viewed with phase optics. The putative early mitotic cells (metaphase) are lo-12 pm in diameter and have a cell surface that is adorned with numerous flattened tubular projections (Fig. 4A). These projections are intermediate in both size and morphology between microvilli and small blebs. Long extensions (retraction fibers21 radiate from these cells and contact the culture dish (Fig. 4A). Some of these processes are branched near their tips. Cells classified as putative telophase cells occur in pairs, often connected by a cylindrical stalk (Fig. 4A). Although their cell surface is often smoother than the ‘early mitotic cell’ they retain variable numbers of surface projections characteristic of the latter, including the radiating processes. Early aggregation stage (non-fusion medium). Early aggregation stage colonies predominate in 11-12-day cultures. Some 60-70% of the cells in these colonies are rounded myoblasts. The central regions of these colonies have aggregates composed of rounded myoblasts. The size of these aggregates is variable and is dependent on colony size and culture age. The peripheral regions of the colonies consist of putative mitotic stages, bipolars, and unaggregated rounded myoblasts. Figure 5 is a phase contrast micrograph of a living aggregation stage colony. We wish to stress that the distinctive rounded morphology of prefusion lizard myoblasts cannot be attributed to an artifact of fixation. This morphology is present in living cultures and

colony containing large numbers of rounded G, prefusion myoblasts. One is shown at higher magnification in Fig. 3. x 512. Bar = 50 pm. FIG. 3. Rounded G, prefusion myoblasts (rm) exhibit a highly blebbed surface topography. Numerous long fdopodia (arrow) are associated with these cells. Also present in the colony are flattened irregularly shaped cells (0. x 2060 bar = 10 pm. Inset (A), rounded cell; x 4000 bar = 5 pm.

group of these cells (rectangle)

FIG. 4. Cell surface topography of mitotic, rounded, and bipolar cells. (A) A putative metaphase cell (m) and a late telophase pair (t). x 2075. Bar = 5 pm. (B) Filopodia from a rounded cell contacting the substratum. Note the terminal swellings (arrows) at the tips of the filopodia. x 5760. Bar = 2 pm. (C) Base of tilopodium (arrow) from a rounded cell, the base of the filopodium is similar in size to the surrounding blebs, x 10,000. Bar = 1 pm. (D) Tip (arrow) of filopodium from one rounded cell contacting a surface bleb of another rounded cell. x 19,000. Bar = 1 pm. (E) Type A bipolar cell typified by a relatively smooth surface topography. A ruffled membrane is present at one pole of the cell. x 1300. Bar = 10 pm. (F) Type B bipolar cell. This cell is shorter and thicker than the Type A bipolar and has a more highly blebbed surface. x 2800. Bar = 5 pm. 246

BAYNE

AND

SIMPSON

Lizard Myogenesis

247

were the major colony type seen in 18- to 20-day cultures. Many of the aggregates contained 50 or more rounded myoblasts (Fig. 9). Here the rounded myoblasts pre6). In colonies, where aggregation has oc- sent yet another characteristic surface tocurred, the rounded myoblasts in medium pography. Although one sees occasional size aggregates (lo-15 cells) have a surface cells that have blebs and/or microvilli, topography that is strikingly different most of the cells present a smooth relafrom that seen in preaggregation stage col- tively unadorned cell surface (Fig. 10). The onies. Although these cells remain spheri- microvilli are for the most part restricted cal in shape, the surface blebs are either to the regions of cell-cell apposition. Indiabsent or greatly reduced in number. In vidual rounded myoblasts found between aggregates in the central regions of the their place one sees numerous microvilli. The microvilli are contorted and entan- colonies also tend to exhibit smooth cell gled with each other. En masse they give surfaces. At the same time, smaller aggrethe rounded-up cell a mossy appearance gates in the more peripheral regions of (Figs. 7 and 8). Rounded myoblasts with these colonies are composed predomithis surface topography are found only in nantly of rounded myoblasts having the aggregation stage colonies. While they oc- mossy topography characteristic of medium sized aggregates. cur predominantly in aggregates, individ“*Stretched-back” cells (mononucleated ual cells outside aggregates can also exmuscle cellslnonfusion medium). Long athibit this mossy topography. Such individual cells are generally found in the central tenuated cells, identical in gross cell shape region of these colonies between aggre- to the stretched-back cells seen in the time-lapse movies, were observed in associgates. Many of the rounded myoblasts found in ation with large aggregates in 18-20-day small aggregates (three to five cells) still cultures. Cells of this morphology were exhibit the highly blebbed surfaces charac- seen to arise from rounded myoblasts in teristic of preaggregation stages. Yet some large aggregates in the time lapse studies. of the rounded cells in these small aggre- Previous studies (Cox, 1968; Simpson and gates exhibit the mossy topography. Cells Cox 1972) have shown that in nonfusion of both surface topographies exhibit long media these stretched-back cells eventually exhibit striations. In older cultures filopodia in the small aggregates. We have observed that even in the me- they frequently undergo spontaneous condium size aggregates where the majority tractions (unpublished observations). The surface topography of these mononuof the rounded myoblasts have the mossy topography, some rounded cells (Fig. 8) cleated muscle cells is similar to that of the rounded myoblasts seen in the large aggrehave a mixture of blebs and microvilli. Inspection of Fig. 8 also reveals that some gates, Their surface is smooth and relaof the rounded myoblasts exhibit large ex- tively unadorned. Patches of microvilli panses of smooth surface free of blebs and and small blebs are occasionally present microvilli. This smooth portion of the cell (Fig. 11). surface generally faces away from the cenFusion cultures (fusion medium). To obtral region of the aggregate. The portion of tain multinucleated myotubes 21-day culthe cell surface contacting other cells tures grown in nonfusion medium and havwithin the aggregate still retains the mi- ing large aggregates of rounded myoblasts crovillar projections. and some stretched back cells were Late aggregation stage (non-fusion me- switched to fusion permissive medium. dium). Late aggregation stage colonies Forty-eight hours following the change to

the percentage of rounded cells in a colony is not altered by fixation procedures utilized in SEM studies (compare Figs. 5 and

BAYNE AND SIMPSON

fusion medium, colonies were selected that had large numbers of multinucleated myotubes. At the SEM level the myotubes the same (Figs. 12 and 13) exhibited smooth surface topography that was seen in the unfused, stretched-back cells maintained in nonfusion medium. The same surface topography was observed in myotubes in cultures that had been grown continuously in fusion medium. DISCUSSION

The present study demonstrates that prefusion G, lizard myoblasts progress through a characteristic developmental sequence prior to terminal differentiation. The facts that lizard prefusion G, myoblasts assume a distinct rounded morphology and that they do not appear in cultures until colonies have reached a fairly large size suggest that the prefusion G, period is different from the previous G, periods through which the cell has progressed. Both time-lapse and SEM evidence supports this. The prefusion G, period is not only extended in time, but it is developmentally distinct with regard to cell behavior and cell surface topography. Information derived from time-lapse studyof lizard myogenic colonies compliments and extends previous studies (Cox, 1968; Simpson and Cox 1972) based on the analysis of staged series of fixed cultures. Myoblasts first assuming the rounded morphology vacillate between rounded and plump bipolar morphologies. However, in all situations where rounded cells constitute 70% of the cell population, the rounded morphology is stable. This stabilization of the rounded G, morphology could be dependent on several factors. Evidence is available (Kahn-Bayne, unpublished)

Lizard

Myogemsis

249

suggesting that the stability of the rounded morphology is density dependent and may be related to factors (Doering and Fischman, 1974; Konigsberg and Buckley, 1974) accumulating in the culture medium. Changes in cell behavior associated with aggregation could also be followed by timelapse cinemicrography. As the number of prefusion cells within the population increases so does the number of cell-cell contacts between these cells. Initially these cell contacts result in ruffled membrane paralysis and change in direction of cell movement. As the proportion of rounded G, myoblasts in the colony reaches 70%, however, many of these cell-cell contacts result in the two cells remaining together and moving in concert. This change in contact behavior presages the formation of small aggregates of rounded prefusion cells. With time more and more rounded prefusion cells are incorporated into these aggregates. In the course of the time-lapse study, rounded prefusion G, cells were never observed to undergo cell division. This observation supports previously published data on lizard myogenesis (reviewed in Simpson and Cox, 1972) suggesting that myoblasts withdraw from the proliferative pool prior to their differentiation as mononucleated or multinucleated muscle cells. This withdrawal from the mitotic cycle is consistent with at least portions of the SO called “quantal” theory of myogenesis (Holtzer and Bischoff, 1970). Although our lizard myoblasts withdraw from the proliferative pool prior to fusion, we would not wish to generalize this phenomenon to encompass all myogenic systems. The results of several studies based on in vitro differen-

FIG. 5. Phase contrast micrograph of a living early aggregation stage colony. In addition to the aggregates of rounded cells (a), individual rounded cells (r), bipolar cells (b), and flattened cells (f) are present. The same relative numbers of the different cell morphologies can be seen in a SEM preparation of an early aggregation colony (Fig. 6). x 500. Bar = 50 pm. FIG. 6. Central region of an carry aggregation colony comparable to the colony illustrated in Fig. 5 but viewed with the SEM. Labels are the same as in Fig. 5. x 500. Bar = 50 pm.

BAYNE

AND

SIMPSON

tiation of avian thigh and breast muscle are clearly incompatible with withdrawal from the cell cycle prior to fusion (O’Neill 1972a; Stockdale and and Stockdale, O’Neill, 1972; Buckley and Konigsberg, 1974; for opposing view see Holtzer et al. (1975)). The withdrawal of large numbers of myoblasts from the cell cycle prior to fusion may be characteristic of muscle having a somite lineage. This suggestion is consistent with the observations of Okazaki and Holtzer (1966) and has been alluded to previously by Patterson and Strohman (1972). The withdrawal of prefusion G, lizard myoblasts (derived from tail regenerates) from the cell cycle may reflect their lineage from somitic mesoderm. Our SEM studies demonstrate that prefusion G1 lizard myoblasts in culture undergo a series of changes in surface topography prior to their differentiation as mononucleated or multinucleated muscle cells. Regardless of whether the cells were cultured in nonfusion medium or fusion medium, they exhibited identical surface features at the level of the SEM. In the lizard system fusion medium acts solely to provide a Ca2+ ion concentration compatible with fusion (Cox and Gunter, 1973). The changes in cell surface topography that we have observed are not related to the Ca2+ ion concentration of the culture medium. While the cell surface changes associated with the fusion of myoblasts are well documented (Kelly and Zacks, 1969; Shimada, 1971; Lipton and Konigsberg, 1972; Fambrough et al., 1974; Rash and Staehelin, 19741, surface changes occurring in prefusion G1 myoblasts prior to and distinct from the fusion event have received less attention.

Lizard

Myogenesis

251

Rounded prefusion G1 lizard myoblasts in young colonies present a highly blebbed cell surface. This characteristic surface topography is shared only with the plump bipolar cells. Both cell types are probably G, cells. The rounded cells are known to be in G, (Cox and Simpson, 1970) and they vacillate initially between the rounded and plump bipolar morphology. This highly blebbed surface topography has been previously ascribed to cultured cells in G, (Porter et al., 1973) and may therefore be indicative of position in the cell cycle. Although the position in the cell cycle could not be ascertained, Shimada (19721, in an SEM study, has also described typical bipolar chick myoblasts as having perinuclear surface blebs. The functional significance of the filopodial and microvillar projections exhibited by the rounded G, myoblasts cannot be assessed on the basis of this study. The role of filopodia and microvilli in in vitro situations is highly speculative. They have been suggested to be concerned in selective attachment of cells (Taylor and Robbins, 1963; McCombs et al., 1968); phagocytosis (Lockwood and Allison, 1966); cell substratum adhesion (Taylor and Robbins, 1963); and Rajaraman et al., 1974); cell aggregation (Pethica, 1961; Lesseps, 1963; Weiss, 1967); cell coupling (DeHaan et al., 1971) and cell fusion (Harris et al., 1966).3 The 3 Dalen and Scheie (1969) have suggested that slender microextensions, similar to the filopodia seen by others and by us in this study, are retraction tibrils. Supposedly they result when a cell pulls in from an extended form in preparation for mitosis. Generally retraction fibrils are devoid of cytoplasmic filaments. Preliminary examination of our material with the TEM, revealed filamentous material in our filopodia and/or microvilli.

FIG. 7. An aggregate of rounded G, prefusion myoblaste from an early aggregation stage colony. The majority of the rounded cells within the aggregate exhibit large numbers of microvilli which give the cell surfaces a “mossy” appearance. Note that most of the individual rounded cells outside the aggregate also exhibit this same surface topography. x 1242. Bar = 10 pm. FIG. 8. Higher magnification of aggregate illustrated in Fig. 7. Although the prefusion cells in aggregation stage colonies remain rounded, surface blebs are absent or greatly reduced in number. (Compare with cell surfaces of prefusion myoblasts in preaggregation colonies Fig. 3.) The prefusion cell labeled (A) exhibits an expanse of smooth cell surface as well as areas covered with microvilli. x 4730. Bar = 2 pm.

BAYNE AND SIMPSON

filopodia and microvilli observed in this study could be involved in any one or all of these proposed functions. We favor the possibility that they are related to the aggregation of the G, myoblasts. We do so for the following reasons: (1) Just prior to overt aggregation the long filopodia processes predominate over the shorter microvilli and interconnect the G, myoblasts; (2) in young (small) aggregates where the distance between cells is reduced, there is a reduction in the number of long filopodia and an increase in the number of short microvilli which then interconnect G, myoblasts within the aggregates; and (3) in both young (small) and older (large) aggregates even cells that possess a very smooth cell surface still exhibit microvilli in areas where cell-cell contact occurs within the aggregate. Our suggested involvement of the filopodia in cell recognition and aggregation is not a new one. Filopodia of thicker dimension than the ones we describe have been suggested to play a role in both cell recognition and aggregation during sea urchin development (Gustafson and Wolpert, 1963) and during sponge cell aggregation (Sindelar and Burnett, 196’7). To date two SEM studies dealing with cell surface changes during cell aggregation have appeared in the literature. Rossomando et al. (1974) working with the cellular slime mold, Dictyostelium, have shown that the cell surfaces of stationary, logphase, and aggregating cells exhibit characteristic topographies. Shimada et al. (1974) have observed the aggregation of dissociated embryonic chick heart cells. In this case aggregation of the dissociated cells was effected via the rotation method

Lizard

Myogenesis

253

of Moscona (1952). Surface specializations (ruffles and microvilli) occurring at sites of intercellular contact were described. Although we choose to relate most of the changes in cell surface topography to the aggregation process, some of the observed changes may be specifically related to the fusion process. From previous studies we know that the rounded G, myoblasts in aggregates undergo a change in shape, from rounded to attenulated-bipolar, prior to the appearance of multinucleated myotubes (Cox, 1968; Simpson and Cox, 1972). This shape change occurs even in media that does not permit fusion. The formation of the long bipolar cells has also been observed in the time lapse movies in this study. SEM observations of these stretched-out myoblasts reveals a surface topography very similar to that of the aggregated rounded cells which give rise to them. Multinucleated myotubes exhibit a similar surface topography. Thus this relatively smooth, unadorned surface topography may be characteristic of cells that are capable of undergoing fusion. A similar cell surface morphology has been described for myoblasts and myotubes in fusing cultures of chick muscle (Shimada, 1972). The present study, although primarily descriptive, serves to define for the first time a discrete set of changes in both cell behavior and surface topography occurring in postmitotic prefusion myoblasts. These changes are related to the appearance, aggregation and eventual differentiation of lizard myogenic cells in uitro. Further studies will be required to define the functional significance of the different surface topographies.

FIG. 9. Central region of a colony at the late aggregation stage. Large aggregates of the G, prefusion cells are present. Compared to the rounded cells seen in early aggregation colonies (see Fig. 8) the majority of the rounded cells here exhibit a characteristic smooth, unadorned cell surface. The aggregate within the square is shown at higher magnification in Fig. 10. x 484. Bar = 50 pm. FIG. 10. Surface detail of prefusion cells within the aggregate indicated in Fig. 9. Although most prefusion cells present a very smooth surface topography, occasional cells with small numbers of blebs and microvilli are also seen. Microvilli (arrow) are still observed between these smooth surfaced cells. x 1767. Bar = 10 pm.

254

DEVELOPMENTAL BIOLOGY

VOLUME 47, 1975

muscle cells (s) have FIG. 11. Like rounded cells in late aggregation stage colonies, “stretched-back” relatively smooth surfaces. Two rounded myoblasts (r) which have not stretched back onto the substratum are also seen. X 1767. Bar = 5 pm. FIGS. 12 and 13. Myotubes from colonies cultured in fusion media. Note the smooth, relatively unadorned cell surfaces similar to those seen on rounded myoblasts in late aggregation stage colonies and on “stretchedback” myoblasts. x 1767. Bar = 5 pm.

BAYNE AND SIMPSON The authors thank P. Forsyth nical assistance with the SEM.

for his expert tech-

REFERENCES ANDERSON, T. F. (1951). Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. Ser. ZZ 13, 130-134. BISCHOFF, R. (1970). The myogenic stem cell in development of skeletal muscle. In “Regeneration of Striated Muscle and Myogenesis” (A. Mauro, S. A. Shtiq, and A. T. Milhorat, eds.), pp. 218-231. Excerpta Medica, Amsterdam. BISCHOFF, R., and HOLTZER, H. (1969). Mitosis and the process of differentiation of myogenic cells in vitro. J. Cell. Biol. 41, 188-200. BUCKLEY, P. A., and KONIGSBERG, I. R. (1974). Myogenic fusion and the duration of the postmitotic gap (G,). Develop. Biol. 37, 193-212. CHLEBOWSKI, J. S., PRZYBYLSKI, R. J., and Cox, P. G. (1973). Ultrastructural studies of lizard (Anolis carolinensis) myogenesis in vitro. Develop. Biol. 33, 80-99. COX, P. G. (1968). In vitro myogenesis of promuscle cells from the regenerating tail of the lizard, Anolis carolinensis. J. Morphol. 126, 1-18. Cox, P. G., and GUNTER, M. (1973). The effect of calcium ion concentration on myotube formation in vitro. Exp. Cell Res. 79, 169-178. Cox, P. G., and SIMPSON, S. B., JR. (19701. A microphotometric study of myogenic lizard cells grown in vitro. Develop. Biol. 23, 433-443. DALEN, H., and SCHEIE, P. (1969). Microextensions on Chang’s liver cells as observed throughout their division cycle. Exp. Cell Res. 57.351-358. DEHAAN, R. L., DUNNING, J. O., HIRAKOW, R., Mo DONALD, T. F., RASH, J., SACHS, H., WILDES, J., and WOLF, H. K. (1971). Carnegie Inst. Washington Yea&. 70, 66-84. DOERING, J. L., and FISCHMAN, D. A. (1974). Characterization of fusion-promoting muscle conditioned media. J. Cell Biol. 63, 86a. FAMBROUGH, D. M., HARTZELL, H. C., POWELL, J. A., RASH, J. E., and JOSEPH, N. (1974). On the differentiation and organization of the surface membrane of a postsynaptic cell-the skeletal muscle fiber. In “Synaptic Transmission and Neuronal Interaction (M. V. L. Bennett, ed.), pp. 285-313. Raven Press, New York. FOLLETT, A. E. C., and GOLDMAN, R. D. (1970). The occurrence of microvilli during spreading and growth of BHK 21/C13 fibroblasts. Exp. Cell Res. 59, 124-136. GUSTAFSON, T., and WOLPERT, L. (1963). The cellular basis of morphogenesis and se’s urchin development. Znt. Reu. Cytol. 15, 139-214. HARRIS, H., WATKINS, J. F., FORD, C. E., and SCHOEFL, G. (1966). Artificial heterokaryons of

Lizard

Myogenesis

255

animal cells from different species. J. Cell Sci. 1, I-30. HOLTZER, H., and BISCHOFF, R. (1970). Mitosis and myogenesis. In “The Physiology and Biochemistry of Muscle as a Food, 2” (E. J. Briskey, R. G. Cassens, and B. B. Marsh, eds.), pp. 29-51. The University of Wisconsin Press, Madison. HOLTZER, H., and SANGER, J. W. (1972). Myogenesis: old views rethought. In “Research in Muscle Development and the Muscle Spindle” (B. Q. Banker, R. J. Przybylski, J. B. Van Der Muelen, and M. Victor, eds.), pp. 122-133. Excerpta Medica, Amsterdam. HOLTZER, H., STRAHS, K., and BIEHL, J. (1975). Thick and thin filaments in postmitotic, mononucleated myoblasts. Science. 188, 943-945. KELLY, A. M., ~~~ZACKS, S. I. (1969). The histogenesis of rat intercostal muscle. J. Cell Biol. 42:135153. KONIGSBERG, I. R. (1963). Clonal analysis of myogenesis. Science. 140, 1273-1284. KONIGSBERG, I. R., and BUCKLEY, P. A. (1974). Regulation of the cell cycle and myogenesis by cellmedium interaction. In “Concepts of Development” (J. Lash and J. R. Whittaker, eds.), pp. 179-193. Sinauer Associates, Inc. Stamford, Connecticut. LASH, J. W., HOLTZER, H., and SWIFT, H. (1957). Regeneration of mature skeletal muscle. Anat. Rec. 128, 679-693. LESSEPS, R. J. (1963). Cell surface projections: Their role in the aggregation of embryonic chick cells as revealed by electron microscopy. J. Exp. Zool. 153, 171-182. LIPTON, B. H., and KONIGSBERG, I. R. (1972). A tinestructural analysis of the fusion of myogenic cells. J. Cell Biol. 53.348-364. LOCKWOOD, W. R., and ALLISON, F. (1966). Electron microscopy of phagocytic cells. III. Morphological findings related to adhesive properties of human and rabbit granulocytes. Brit. J. Ezp. Pathol. 47, 158-162. MCCOMBS, R. M., BENYESH-MELNICK, M., and BRUNSCHWIG, J. P. (1968). The use of millipore filters in ultrastructural studies of cell cultures and viruses. J. Cell Biol. 36, 231-243. MINTZ, B., and BAKER, W. W. (1967). Normal mammalian muscle differentiation and gene control of isocitrate dehydrogenase synthesis. Proc. Nat. Acad. Sci. USA 58, 592-598. MOSCONA, A. A. (1952). Cell suspensions from organ rudiments of chick embryos. Exp. Cell Res. 3,535539. OKAZAKI, K., and HOLTZER, H. (1966). Myogenesis: fusion, myosin synthesis, and the mitotic cycle. Proc. Nat. Acad. Sci. USA. 56, 1484-1490. O’NEILL, M. C., and STOCKDALE, F. E. (1972a). A kinetic analysis of myogenesis in vitro. J. Cell Biol. 52, 52-65.

256

DEVELOPMENTAL

BIOLOGY

O’NEILL, M. C., ~~~STOCKDALE, F. E. (1972b). Differentiation without cell division in cultured skeletal muscle. Develop. BioZ. 29, 410-418. PATTERSON, B., and STROHMAN, R. C. (1972). Myosin synthesis in cultures of differentiating chicken embryo skeletal muscle. Develop. Biol. 29, 113138. PF,THICA, B. A. (1961). The physical chemistry of cell adhesion. Exp. Cell Res. (Suppl.) 8, 123-140. PORTER, K. R., KELLEY, D., and ANDREWS, P. M. (1972). The preparation of cultured cells and soft tissues for scanning electron microscopy. In “Proceedings at the Fifth Annual Stereoscan Colloquium-1972,” pp. 1-19. PORTER, K., PRESCOTT, D., and FRYE, J. (1973). Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J. Cell BioZ. 57:815-836. RAJARAMAN, R., ROUNDS, D. E., YEN, S. P. S., and REMBAUM, A. (1974). A scanning electron microscope study of cell adhesion and spreading in vitro. Exp. Cell Res. 88, 327-339. RASH, J. E., and FAMBROUGH, D. (in consultation with J. D. Ebert). (1970). Further studies of factors regulating the fusion of myoblasts. Preliminary observations of fusion in developing rat skeletal muscle. Carnegie Inst. Washington Yearb 70, 64-65. RASH, J. E., and STAEHELIN, L. A. (1974). Freezecleave demonstration of gap junctions between skeletal myogenic cells in uiuo. Develop. BioZ. 36, 455-461. ROSSOMANDO, E. F., STEFFEK, A. J., MUJWID, D. K., and ALEXANDER, S. (1974). Scanning electron microscopic observations on cell surface changes during aggregation of Dictyostelium discoidium. Exp. Cell Res. 85, 73-78. Shimada, Y. (1971). Electron microscope observations on the fusion of chick myoblasts in uitro. J. Cell BioZ. 48, 128-142.

VOLUME

47,

1975

Y. (1972). Scanning electron microscopy of myogenesis in monolayer culture: A preliminary study. Develop. BioZ. 29, 227-233. SHIMADA, Y., MOSCONA, A. A., and FISCHMAN, D. A. (1974). Scanning electron microscopy of cell aggregation: cardiac and mixed retina-cardiac cell suspensions. Develop. BioZ. 36, 428-446. SIMPSON, S. B., JR., and Cox, P. G. (1972). Studies on lizard myogenesis. In “Research in Muscle Development and the Muscle Spindle” (B. Q. Banker, R. J. Przybylski, J. B. Van Der Muelen, and M. Victor, eds.), pp. 72-87. Excerpta Medica, Amsterdam. SINDELAR, W. F., and BURNETT, A. L. (1967). A timelapse photographic analysis of sponge cell-reaggregation. J. Gen. Physiol. 50, 1089-1090. STOCKDALE, F. E., and O’NEILL, M. C. (1972). Deoxyribonucleic acid synthesis, mitosis, and skeletal muscle differentiation. In Vitro 8, 212-225. STREHLER, B. L., KONIGSBERG, I. R., and KELLEY, J. E. T. (1963). Ploidy of myotube nuclei, developing in uitro as determined with a recording doublebeam microspectrophotometer. Exp. Cell Res. 32, 232-241. TAYLOR, A. C. (1966). Microtubules in the microspikes and cortical cytoplasm of isolated cells. J. Cell BioZ. 28, 155-168. TAYLOR, A. C., and ROBBINS, E. (1963). Observations on microextensions from the surface of isolated vertebrate cells. Deuelop. BioZ. 7, 660-673. WEISS, L. (1967). “The Cell Periphery, Metastasis, and Other Contact Phenomena.” North-Holland, Amsterdam. YAFFE, D. (1971). Developmental changes preceding cell fusion during muscle differentiation in vitro. Exp. Cell Res. 66, 33-48. YAFFE, D., and FELDMAN, M. (1965). The formation of hybrid multinucleated muscle fibers from myoblasts of different genetic origins. Develop. BioZ. 11,300-317. SHIMADA,